Dimethylpolysiloxane suspensions of biologics and preparation thereof

ABSTRACT

A STABLE SUSPENSION OF A BIOLOGICAL IN DIMETHYLPOLYSILOXANE IS PROVIDED CONTAINING ONE OR MORE POLYHYDRIC ALCOHOLS. EXEMPLARY OF SUCH STABLE SUSPENSIONS IS A SUSPENSION CONTAINING SMALLPOW VACCINE, DIMETHYL POLYSILOXANE, 4 TO 10% BY WEIGHT MANNITOL, AND 4 TO 10% BY WEIGHT SORBITOL. THE BIOLOGICAL SUSPENSIONS MAINTAIN THEIR EFFICACY, POTENCY, AND SUSPENSION CHARACTERISTICS UNDER PROLONGED STORAGE AND CAN BE DIRECTLY WITHDRAWN FROM THEIR CONTAINERS AND ADMINISTERED TO THE PATIENT.

United States Patent 3,577,524 DIMETHYLPOLYSILOXANE SUSPENSIONS 0F BIOLOGI'CS AND PREPARATION THEREOF Walter David Pratt, Spring Valley, NY, assignor to American Cyanamid Company, Stamford, Conn. No Drawing. Filed Mar. 4, 1968, Ser. No. 709,940

Int. Cl. C12k 5/00 U.S. Cl. 424-89 8 Claims ABSTRACT OF THE DISCLOSURE A stable suspension of a biological in dimethylpolysiloxane is provided containing one or more polyhydric alcohols. Exemplary of such stable suspensions is a suspension containing smallpox vaccine, dimethyl polysiloxane, 4 to 10% by weight mannitol, and 4 to 10% by weight sorbitol. The biological suspensions maintain their efiicacy, potency, and suspension characteristics under prolonged storage and can be directly withdrawn from their containers and administered to the patient.

This invention relates to stable suspensions of biologicals. More particularly, it relates to stable, dimethylpolysiloxane suspensions of vaccines containing polyhydric alcohols, and to a method for preparing these sus pensions.

While this invention relates generally to stable suspensions of a variety of biological compositions, including vaccines, and the like, it is particularly applicable to stable suspensions of vaccines. The invention, therefore, will be described as it relates to vaccines but it is to be understood that it is applicable to all such biological compositions.

Vaccines generally consist of killed microorganisms, attenuated microorganisms, or live, viral microorganisms, such as bacteria, rickettsia, or virus. These vaccines in clude the production of antibodies within the body of the person or animal treated with them to provide immunity against the specific microorganisms used in the vaccine. All of such vaccines, however, rapidly lose their potency when stored at room temperature for prolonged periods of time. Viral vaccines are particularly subject to loss of potency on prolonged storage.

While storage of vaccines under refrigeration is helpful in increasing their span of effectiveness, the problems inherent in the shipment and storage of the vaccines under refrigeration has greatly hindered their successful and economical use in large-scale immunization programs. This is especially troublesome in underdeveloped areas of the world where disease is prevalent and refrigeration facilities are generally inadequate or not available.

Because of the problems encountered in the shipment and storage of vaccines, various proposals have been advanced for the provision of vaccine compositions that do not lose their potency during storage even in the absence of continuous refrigeration. In one recently developed method the vaccine is suspended in a substantially anhydrous medium that protects it from contact with moisture.

'It is, of course, important that the protective medium used does not itself adversely affect the potency of the vaccine, and dimethylpolysiloxane is the preferred media for use in this method because it not only provides a storage stable vaccine and does not adversely affect the potency of the vaccine, but can be safely administered to the patient along with the vaccine. This latter characteristic permits the dimethylpolysiloxane to be retained as the vaccines diluent.

Vaccines suspended in dimethylpolysiloxane can be prepared by first drying the vaccine and then mixing the dried vaccine with the dimethylpolysiloxane to form a suspen- 3,577,524 Patented May 4, 1971 sion. While the vaccine suspension is storage-stable and does not require the use of refrigeration to maintain the efiicacy and potency of the vaccine, its suspension characteristics are not stable and previously available vaccine suspensions tend to separate out upon standing.

'In the past, therefore, the administrator of such a vaccine composition had to physically mix or shake the composition to resuspend each dose of vaccine before it could be administered to the patient. Obviously, and especially in the case of large-scale immunization programs, the necessity of resuspending each dose of vaccine greatly increases the effort and time required to successfully complete the immunization program.

It is therefore a primary object of this invention to provide dimethylpolysiloxane suspensions of biologicals having permanent suspension characteristics.

Another object of this invention is to provide stable suspensions of vaccines in dimethylpolysiloxane that maintain their efficacy and potency under prolonged storage and that can be safely and directly administered to the patient.

Yet another object of this invention is to provide a novel class of suspending agents for producing safe, potent, and stable suspensions of viral vaccines in dimethylpolysiloxane.

Still another object of this invention is to provide an improved process for producing stable suspensions of biologicals in dimethylpolysiloxane.

Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention, the objects and advantages being realized and attained by means of the compositions, processes, and improvements particularly pointed out in the appended claims.

To achieve the foregoing objects and in accordance with its purpose, this invention, as embodied and broadly described, provides a stable, biological suspension comprising a biological, dimethylpolysiloxane, and from about 4.0 to 10.0% by Weight of a polyhydric alcohol selected from the group consisting of mannitol, dulcitol, and inositol.

The invention further provides a process for preparing a stable, biological suspension which comprises suspending in dimethylpolysiloxane, a dry mixture containing a biological and a polyhydric alcohol selected from the group consisting of mannitol, dulcitol, and inositol, the biological suspension containing from about 4 to 10% by weight of the alcohol.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory but are not restrictive of the invention.

The dimethylpolysiloxanes that are useful in preparing the vaccine suspensions of this invention are based on the following repeating structural unit:

in which each R is methyl.

The dimethylpolysiloxanes that are useful in the suspension compositions of this invention include straightchain and cross-linked polymers having a wide range of molecular weights. The lower molecular weight, straightchain polymers are preferred for use in this invention because they are liquids, and have satisfactory viscosities for producing effective vaccine suspension compositions. These lower molecular weight polysiloxanes generally have inherent viscosities of from about 0.65 to 1 million centistokes at 25 C. Polysiloxanes having an inherent pared by mixing an alcohol with the vaccine prior to suspending the vaccine in the polysiloxane. The alcohol serves as a suspending agent for the vaccine to provide a permanent suspension without affecting the potency, efficacy, or safety of the vaccine compositions.

The alcohols useful in this invention are mannitol, dulcitol and inositol. These alcohols can be used either individually or in the form of a mixture of two or more of them. It is to be understood, therefore, that the term alcoho as used throughout this specification and the appended claims, is intended to refer to any one or a mixture of two or more of these alcohols.

While mannitol, dulcitol, and inositol, alone or in ad mixture, provide stable suspensions of vaccines in dimethylpolysiloxane in accordance with this invention, it has been found desirable to also include certain amounts of sorbitol, in such suspensions to achieve optimum suspension characteristics. Sorbitol is known to have a stabilizing effect on the potency of vaccines, but in addition, it unexpectedly has been found to enhance the stabilization of the suspension compositions of this invention when used in combination with one or more of mannitol, dulcitol and inositol. It is to be understood that sorbitol Will not provide a stable vaccine suspension if it is used alone and it is intended to be used only in combination with the other alcohols described above in achieving an optimum vaccine suspension. An equal mixture by weight of sorbitol and one or more of these alcohols is preferred for use in the suspension compositions of this invention.

The stable, dimethylpolysiloxane suspensions of this invention are prepared in such proportions that the final suspension product contains from about 4.0 to 10.0% by weight of one of the above alcohols based on the volume of the suspension and the dry weight of the alcohol. Thus, for example, a dried vaccine suspended in 100 ml. of dimethylpolysiloxane should contain from 4.0 to 10.0 grams of mannitol, dulcitol and/ or inositol. When the suspension contains from about .04 to .10 gram of the alcohol per ml. of suspension, therefore, the amount of alcohol in such a suspension, is said to be 4.0 to 10.0% by Weight, and it will be understood that the term by weight is referred to in this manner throughout this specification and the appended claims.

In a preferred embodiment of this invention the dimethylpolysiloxane suspension contains from about 4 to by weight mannitol and from about 4 to 10% by weight sorbitol.

Any suitable procedure can be employed to prepare the vaccine used in the suspensions of this invention. For example, the microorganisms can be obtained from virus strains, or directly from an animal or human patient and propagated in chick embryo tissue, tissue culture fluids, or any other suitable media. Exemplary of such vaccines is smallpox virus grown on the membranes of smallpoxinfected embryonated eggs.

In accordance with a preferred embodiment of this invention, a fluid vaccine is mixed with an aqueous solution of one of the alcohols, either alone or in admixture with sorbitol in such proportions that the resulting mixture contains from about 4 to 10% by weight of the alcohol and 4 to 10% by Weight of sorbitol, if present. The fluid mixture of vaccine and alcohol is throughly blended and then vacuum-dried to a uniform cake.

The dry mixture is then broken up and a milling agent, such as glass beads or stainless steel balls, is added together with an amount of the dimethylpolysiloxane suffi cient to produce a suspension having a volume approximately equal to the original volume of the fluid mixture of vaccine and alcohol before it was dried. Milling is continued until a uniform suspension of the vaccine in the dimethylpolysiloxane is obtained.

The dimethylpolysiloxane-vaccine suspensions of this invention containing one or more of mannitol, dulcitol and inositol as a suspending agent, either alone or in combination with sorbitol, have permanent suspension characteristics in which the vaccine does not substantially separate from the liquid phase of the dimethylpolysiloxane under prolonged periods of storage. Further, the vaccine suspensions of this invention maintain their etficacy and potency under storage and can be withdrawn from their container and directly and safely administered to the patient.

For a clearer understanding of this invention, specific examples of it are set forth below. There examples illustrate the preparation of the dimethylpolysiloxane vaccine suspensions of this invention.

The examples, however, are merely illustrative and are not to be understood as limiting the scope and underlying principles of the invention in any way.

EXAMPLE 1 Smallpox virus is grown on the membranes of smallpoxinfected embryonated eggs. The allantoic membranes containing live smallpox virus are harvested and homogenized. The homogenized vaccine is then mixed with an equal volume of a aqueous solution containing 18.2% by Weight mannitol and 18.2% by weight sorbitol. Thus 50 ml. of an aqueous solution containing 9.1 grams of mannitol and 9.1 grams of sorbitol is mixed with every 50 ml. of fluid vaccine so that the resulting fluid mixture of vaccine, mannitol, and sorbitol contains 9.1% by weight mannitol and 9.1% by weight sorbitol. The mixture is separated into ml. samples, and each sample is vacuum-dried to a uniform cake.

Each cake is partially broken and stainless steel balls are added to it along with 100 milliliters of dimethylpolysiloxane (viscosity of 350 cs. at 25 C.). Milling is continued until a uniform suspension of the smallpox vaccine in the organopolysiloxane is obtained.

After milling, the stainless steel balls are removed and the resulting vaccine suspensions are stored in a sealed container for six (6) months at 24 C. At the end of this period, the suspension characteristics of the vaccine composition remain unchanged. The activity of the vaccine at the end of the storage period is measured by performing potency tests on the chloroallantoic membrane of chick embryo in terms of pock-forming units per milliliter. The potency of the vaccine is found to remain unchanged even after the prolonged storage period.

EXAMPLE 2 The procedure of Example 1 is repeated in this example using 5.5% by weight sorbitol, in admixture with 9.1% by weight mannitol as the suspending agent for the vaccme.

The potency of the vaccine and the suspension characteristics of the vaccine composition again remain unchanged after a six-month storage period.

EXAMPLE 3 The procedure of Example 1 is repeated in this example using 4.0% by weight dulcitol in place of the mixture of mannitol and sorbitol used in Example 1.

The suspension characteristics of the vaccine compositions changed by only about 3%, as measured by each samples ratio of clear dimethylpolysiloxane to suspension after the six-month storage period.

EXAMPLE 4 The procedure of Example 1 is repeated, using 9.1% by weight mannitol in place of the mixture of mannitol and sorbitol used in Example 1.

The suspension characteristics of the vaccine compositions changed by only about 2% after the six-month storage period.

EXAMPLE 5 The procedure of Example 1 is repeated, using 9.1% by weight sorbitol in place of the mixture of mannitol and sorbitol in Example 1.

The suspension characteristics of the vaccine compositions changed by only about 8% after the six-month storage period.

EXAMPLE 6 This example illustrates the stabilizing eflfect achieved by the class of alcohols of this invention on vaccines suspended in organopolysiloxanes, in comparison with suspensions containing only sorbitol,

The procedure of Example 1 is repeated using 9.1% by weight sorbitol in place of the mixture of mannitol and sorbitol in Example 1.

The suspension characteristics of the vaccine compositions changed by about 48% after the six-month storage period. This is approximately equivalent to the suspension characteristics of an organopolysiloxane vaccine suspension containing no suspension-improving agent.

The invention in its broader aspects is not limited to the specific details shown and described and departures may be made from such details within the spirit and scope of the accompanying claims without departing from the principles of the invention and Without sacrificing its chief advantages.

What is claimed is:

1. A process for preparing a uniform, stable, live virus vaccine suspension having permanent uniform suspension characteristics so that the suspension does not have to be physically mixed or shaken to suspend each dose of vaccine prior to administration to a patient, which comprises admixing in dimethylpolysiloxane, a dry mixture of a live virus vaccine and a polyhydric alcohol selected from the group consisting of mannitol, dulcitol, and inositol to provide a vaccine suspension containing from about 4.0 to 10.0% by weight of the alchohol, said live virus vaccine not substantially separating from the liquid phase of dimethylpolysiloxane after a six-month period of storage, whereupon it can be withdrawn from a sealed container and administered directly to the patient without resuspension.

2. The process of claim 1, in which the mixture of vaccine and alcohol also includes sorbitol, the vaccine suspension containing from about 4.0 to 10.0% by weight of sorbitol.

3. The process of claim 2, wherein the vaccine suspension includes from about 4.0 to 10.0% by weight mannitol and from about 4.0 to 10.0% by weight sorbitol.

4. The process of claim 1, wherein the vaccine is smallpox vaccine.

5. A stable, live virus vaccine suspension having permanent suspension characteristics comprising a live virus vaccine, dimethylpolysiloxane, and from about 4.0 to 10.0% by weight of at least one polyhydric alcohol selected from the group consising of mannitol, dulcitol and inositol, prepared in accordance with the process of claim 1.

6. The suspension of claim 5, which includes from about 4.0 to 10.0% by weight sorbitol in admixture with the polyhydric alcohol.

7. The suspension of claim 6, wherein the suspension includes a mixture of from about 4.0 to 10.0% by weight sorbitol and from about 4.0 to 10.0% by weight mannitol.

8, The suspension of claim 5, wherein the vaccine is smallpox vaccine.

References Cited UNITED STATES PATENTS 2,879,202 3/1959 Aiston et al. 42489 3,214,340 10/1965 Laurence 424-89 3,378,443 4/1968 Cooper et al. 424--89 SHEP K. ROSE, Primary Examiner US. Cl. X.R. 

